Negative Cooperativity and High Affinity in Chitooligosaccharide Binding by a Mycobacterium smegmatis Protein Containing LysM and Lectin Domains

Abstract

LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides has been underexplored. Binding of a Mycobacterium smegmatis protein containing a lectin (MSL) and one LysM domain to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration that demonstrate the presence of two binding sites of nonidentical affinities per dimeric MSL-LysM molecule. The affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in the MSL-LysM molecule, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the case presented here, two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of Bacillus subtilis and Lactococcus lactis. Also, the affinity of the MSL-LysM molecule for chitooligosaccharides is higher than that of LysM-chitooligosaccharide interactions reported so far.

Multitasking Human Lectin Galectin-3 Interacts with Sulfated Glycosaminoglycans and Chondroitin Sulfate Proteoglycans

“…As Hill plots are logarithmic representations, they are capable of plotting all theoretically obtainable data. Hill plots have been employed to reveal cooperativity associated with binding of multivalent ligands to lectins , When using Hill plot analysis for multivalent binding, the term for the fraction of bound ligand, X b / M t , is corrected for the valency of the sugar to give ( X b ) (functional valency of sugar)/ M t, which is a modification of the classical Hill plot. , A similar modification was done in the present study (see Experimental Procedures).…”

“…Binding of multivalent ligands to lectins has been shown to be associated with progressively increasing negative cooperativity. ,, CSA, CSC, and BPG are multivalent ligands of Gal-3. Hill plot analysis shows that these three ligands bind to Gal-3 with progressively increasing negative binding cooperativity.…”

“…ITC binding data of CSA, CSC, and the bovine proteoglycan were subjected to Hill plot analysis to understand if binding was associated with cooperitivity. , Hill plots were obtained by plotting log­[ Y ( i )/[1 – Y ( i )]] versus log­[ X f ( i )], where Y ( i ) is [ X b ( i )] (functional valency of ligand)/ M t­( i ) [where X b ( i ) = concentration of bound ligand after the i th injection, X f ( i ) = concentration of free ligand after the i th injection, and M t ( i ) = total lectin concentration after the i th injection]. These are modified versions of the Hill plots that accommodate multivalent binding by taking into account the functional valency of the ligand. , ITC-driven functional valencies of CSA, CSC, and the bovine proteoglycan for Gal-3 were used in these calculations.…”

“…ITC binding data of CSA, CSC, and the bovine proteoglycan were subjected to Hill plot analysis to understand if binding was associated with cooperitivity. 38,39 Hill plots were obtained by plotting log…”

“…These are modified versions of the Hill plots that accommodate multivalent binding by taking into account the functional valency of the ligand. 38,39 ITC-driven functional valencies of CSA, CSC, and the bovine proteoglycan for Gal-3 were used in these calculations.…”